1. Crescimento de H. Capsulatum <ol><li> Prepare um gabinete de biossegurança para o trabalho com o fungo com a limpeza do gabinete com lixívia a 10%, seguido por 20 min de irradiação UV. H. capsulatum na fase leveduriforme Biossegurança exige nível práticas (BSL) II, enquanto a forma de moldes requer BSLIII. Use o formulário de leveduras nas etapas seguintes. </li><li> Em seguida preparar um tubo cônico de 15 ml com 5 ml de PBS. Colheita uma colônia de H. capsulatum (linhagem ATCC Hc G217B) de uma placa de agar sangue BHI [glicose a 10 g / L, cisteína 0,1 g / L, a penicilina-estreptomicina 1% e os carneiros células vermelhas do sangue 50 ml / L (Colorado Serum Co., Colorado, EUA) ] cultivadas a 37 ° C ou tomar uma alíquota do estoque de fermento congelado a -80 ° C e adicioná-lo à PBS. Vortex vigorosamente as células e centrifugar para 1100 xg por 10 min em temperatura ambiente. Remova cuidadosamente o sobrenadante sem perturbar o sedimento e adicionar 10ml de PBS fresco. Repita este procedimento três vezes. Suspender as células em 5 mL de PBS e tranfer para um tubo cônico de 50 mL. </li><li> Para perturbar as células agregadas, passe a suspensão de células de levedura por meio de um G1 26 / 2 agulha u cantar uma seringa de 10 mL 5 vezes em uma cabine de segurança biológica (usando proteção facial). Para isolar o fermento, pequeno de tamanho uniforme, centrifugar as células a 55 xg por 1 min em temperatura ambiente e depois remover o top 1 ml. </li><li > Adicione a suspensão celular para um erlenmeyer estéril, contendo 100 ml de meio HAM F12 (GIBCO) suplementado com 16g / L de glicose, 1 g / L de ácido glutâmico, 8.4mg / L cistina, 6g / L de ácido glutâmico e, em seguida, as células crescem a 37 ° C por 48 h em uma incubadora com agitação 150rpm. </li></ol> 2. Hibridoma Crescimento e Purificação de anticorpos monoclonais <ol><li> Transferir os hibridomas selecionados para DMEM contendo 10% de soro fetal bovino, NCTC 10%, 1% não-aminoácidos essenciais e 1% penicilina-estreptomicina. Cultivar as células em um frasco de cultura de células (Becton Dickinson) por 5-7 dias a 37 ° CO C / 5% 2. </li><li> Em seguida, centrifugar a média em 1100 xg por 10 min à temperatura ambiente e recolher todas as sobrenadante sem perturbar as pelotas. </li><li> Em seguida, purificar o sobrenadante livre de células usando uma proteína A / G resina por FPLC. </li><li> A concentração mAb pode então ser calculada por um ELISA de captura (1) usando um controle de isotipo IgG como padrão (Figura 1B). </li></ol> 3. Preparação do inóculo Histoplasma capsulatum <ol><li> Dê uma 48 h Histoplasma capsulatum (Hc estirpe ATCC G217B) cultura fase de levedura cultivada em meio HAM F-12. </li><li> Centrifugar as células a 1100 xg por 10 min. Desprezar o sobrenadante e adicionar PBS fresco. </li><li> Repita o procedimento 3 vezes 3,2. </li><li> Passe a suspensão de células de levedura 5 vezes através de uma agulha 26G1 / 2, utilizando uma seringa. </li><li> Centrifugar a suspensão a 55 xg por 1 min a clusters pellet celular residual. Transferir o sobrenadante contendo a suspensão única célula para um novo tubo. </li><li> Enumerar a suspensão de células usando uma única hematocytometer. </li><li> Ajustar a concentração de células, a fim de atingir 1,25 x 10 7 levedura (sobrevivência) ou 5,0 x 10 6 (CFU, citocinas e histologia) em um Suspenção de &lt;mL 50 (Figura 2). </li></ol> 4. Administração intraperitoneal da MAbs <ol><li> Pegar o rato pelo rabo e da nuca do pescoço (Fig. 3A). Imobilizar o mouse pela nuca de seu pescoço tão perto theears possível (certifique-se de assumir a pele suficiente para que o mouse cannotturn sua cabeça para morder a pessoa manuseá-lo; Figura 3B e 3C). </li><li> Estabilizar o dedo withthe rabinho suavemente pressionado contra a palma da mão (Figura 3D). </li><li> Swab a zona da injecção com etanol 70% antes de colocar a agulha e para aspirar a olhar para o sangue antes de injetar. </li><li> Use o 26G1 / 2 a piercethe pele e abdominalmuscles para injectar a solução mAb contendo 500μg (PBS, isotipo de anticorpo de controle ou o mAb a serem testadas, diluídas em 1 mL PBS) em esquerda thelower ou quadrante inferior direito abdominal (cavidade intraperitoneal), com o animal na cabeça para baixo, tomando cuidado para evitar os órgãos internos andother diafragma (Figura 3E). </li><li> Esperar um pouco antes de retirar o needleto reduzir a probabilidade de vazamentos. </li><li> Espere um mínimo de duas horas antes de prosseguir para a próxima etapa. </li></ol> 5. Anestesia do mouse e Infecções intranasal <ol><li> Prepare a anestesia com ketamina e xilazina a 100 mg / Kg e 10 mg / Kg, respectivamente. Executar a administração intraperitoneal de PBS, isotipo de anticorpo controle, ou anticorpos experimental, conforme descrito no protocolo escrito que o acompanha. Esperar duas horas após a injeção de mAb antes de anestesiar o rato e prosseguir com a infecção intranasal. Prepare anestesia com quetamina e xilazina a 100 mg / Kg e 10 mg / kg, respectivamente (7).<html> </li><li> Durante o período de 2h de espera, prepare o inóculo capsulum Histoplasma, conforme descrito no protocolo escrito que o acompanha. </li><li> Proceder de acordo com os itens 5,1-5,5. Toe pitada os animais com uma pinça e verificar a ausência de reflexo e confirmam que a anestesia funcionou. Anaesthetization Depois, gentilmente suspender o mouse por seus dentes da frente a uma corda de nylon ou algodão. Use 2 está coluna para amarrar as cordas, a fim de suspender os animais. </li><li> Lentamente administrar cerca de 50 mL de inóculo em um nare usando uma micropipeta, enquanto monitorando de perto a taxa de rato respiração. Permitir que o mouse para descansar nesta posição por 2-5 min após a infecção intranasal para facilitar a deposição da levedura nos pulmões do animal (Figura 3F). </li><li> Depois de o rato tenha sido infectado, manter o mouse em um ambiente monitorado quente (temperatura entre 25 e 37 ° C), com cuidado, utilizando uma lâmpada de calor, se necessário, até que ele se recupera da anestesia. Quando o desperta mouse, devolvê-lo para uma gaiola limpa, com acesso ad libitum à água e comida. As gaiolas são devolvidos para as nossas instalações de animais e mantidos em uma sala de animal limpo (BLSI). </li></ol> 6. Estudos de sobrevivência <ol><li> Avaliar os animais infectados e mock-infectados clinicamente por taquipnéia, letargia, obnubilação e perda de peso. Os animais devem ser verificados duas vezes por dia por membros de laboratório e diariamente por guardas Animal. </li><li> Com histoplasmose murina, até perto do fim da vida, normalmente há nenhum sinal aparente de infecção que não seja um ligeiro aumento da taxa de respiração. Com doença avançada, o que geralmente acontece a partir de 10-11 dias, os ratos tornaram-se significativamente taquipnéicos e têm atividade diminuída. Normalmente, eles rapidamente se tornam moribunda e expirar. </li><li> Afligido animais devem ser sacrificados de forma adequada com CO 2 em uma câmara dedicada, falecido animais devem ser enumeradas diária. </li></ol> 7. Estudos Estudos CFU Histologia e Citocinas <ol><li> Prepare 15 ml tubos cônicos de 10 mL com PBS para cada órgão a ser coletado. </li><li> Euthanize animais 7 e 14 dias após a infecção com um inuculum subletais (5,0 x 10 6), como demonstrado anteriormente e remover o baço, pulmão e fígado imediatamente. </li><li> Remover um pedaço do órgão a ser avaliado e realizar a fixação em formol durante a noite. As seções serão examinadas sob o microscópio para avaliação patológica. </li><li> Prepare 50 ml tubos cônicos com PBS 5 mL coberto com 70 filtros de células mm para cada órgão a ser homogeneizados. Macerar cada parcela restante dos órgãos, separadamente para os 50 mL utilizando tubos êmbolos seringa estéril 5mL. </li><li> Em seguida, em série diluir homogeneizados de órgãos (1:100, 1:1000 e 1:5000) e 100μL placa de cada diluição em placas BHI-ágar sangue (2). </li><li> Adicionar comprimidos de inibidor de protease para o homogeneizados de acordo com as instruções do fabricante (Roche, Alemanha). </li><li> Incubar as placas a 37 ° C por até 14 dias e UFC enumerar. </li><li> Centrifugar a homogeneizados de órgãos em 4000xg por 10 min e retire sobrenadantes órgão, whish será usado em usados ​​nos ensaios de citocinas realizada por métodos padronizados de acordo com o fabricante. </li></ol> 8. Proteção anticorpo monoclonal contra H. Capsulatum é dependente e Especificidade de Anticorpos do isotipo <ol><li> O mAb isotipo é fundamental na proteção contra H. capsulatum, como camundongos tratados com IgG1 ou IgG2a mAbs a Hsp60 têm sobrevida significativamente prolongada em comparação com camundongos controle recebendo um mAb IgG2b a Hsp60 ou seja um isotipo controle mAb ou PBS (Figura 4). </li><li> Além disso, em camundongos que receberam anticorpos monoclonais de proteção, houve uma redução significativa no número de CFU pulmonar e esplênica no dia 7 e diminui de 2 e 2,5 unidades log de números de levedura nos pulmões no dia 14 (Figura 5). </li></ol><img src="/files/ftp_upload/2532/2532fig1.jpg" alt="Figura 1" /> Figura 1: Crescimento do H. capsulatum e hibridomas. (A) preparação líquida HAM F-12 cultura a partir de uma única colônia obtida H. capsulatum cultivadas em placas de BHI ágar-sangue. (B) o crescimento Hibridoma em frascos de cultura de células e purificação mAb pela proteína A / G resina cromatografia de afinidade usando. <img src="/files/ftp_upload/2532/2532fig2.jpg" alt="Figura 2" /> Figura 2: H. capsulatum preparação do inóculo para a infecção intranasal. Suspensão de células obtidas após H. interrupção agregados capsulatum por seringa e centrifugação é enumerado pela contagem em um hemocitômetro. Concentração de células é ajustado para 1,25 x 10 7 (experimentos de sobrevivência) ou 5 x 10 6 (UFC, citocinas e histologia) em &lt;mL 50. upload/2532/2532fig3.jpg &quot;alt =&quot; Figura 3 &quot;/&gt; Figura 3: manuseio do mouse durante mAb administração. (A) O mouse é pego pelo rabo e (B e C) imobilizada segurando a nuca de seu pescoço tão perto theears. (D) A cauda é realizada entre o dedo mindinho e na palma. (E) A solução mAb é injetado com uma agulha 26G1 / 2. (F) O H. capsulatum inóculo é administrada por via intranasal com cuidado pipetando suspensão de células em um nare. <img src="/files/ftp_upload/2532/2532fig4.jpg" alt="Figura 4" /> Figura 4: MAbs a Hsp60 pode alterar a patogênese da histoplasmose. injeções ip com 500 mg de IgG1, IgG2a mAbs ou 2 h antes para a sobrevivência de infecção significativamente prolongada (p &lt;0,05, em comparação com controles), mas um IgG2b mAb. <img src="/files/ftp_upload/2532/2532fig5.jpg" alt="Figura 5" /> Figura 5: determinações CFU nos pulmões, 7 e 14 dias após um desafio subletais intranasal com 5x10 6 leveduras Hc de camundongos tratados com ip mAbs selecionados para Hsp60 ou mAb irrelevante mostrou redução na carga fúngica para IgG1 e IgG2a mAbs animais tratados. Barras pretas representam UFC no dia 7 e barras brancas 14 dias pós-infecção (* p &lt;0,001 em 7 dias e ** p &lt;0,001 em 14 dias após a infecção).

<ol>
	<li>Casadevall, A., Mukherjee, J., Scharff, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+based+ELISAs+for+cryptococcal+polysaccharide.">Monoclonal antibody based ELISAs for cryptococcal polysaccharide.</a> J. Immunol. Methods. 154, 27-35 (1992).</li><li>Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+heat+shock+protein+60+alter+the+pathogenesis+of+Histoplasma+capsulatum.+Infect+Immun+77.">Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77.</a> , 1357-1367 (2009).</li><li>Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+function+and+molecular+mapping+of+M+antigen+in+yeast+phase+of+Histoplasma+capsulatum.">Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum.</a> PLoS One. 3, e3449-e3449 (2008).</li><li>Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+and+endemic+dimorphic+fungi.">Protective antibodies and endemic dimorphic fungi.</a> Curr Mol Med. 5, 435-442 (2005).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &amp;amp, G. S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J Clin Invest. 112, 1164-1175 (2003).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J. Clin. Invest. 112, 1164-1175 (2003).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab Anim. 27, 30-39 (1993).</li></ol>

Não há conflitos de interesse declarados.

O protocolo aqui apresentado demonstra que mAb de H. capsulatum pode modificar o curso de histoplasmose murina experimental. MAbs aos antígenos de superfície celular de patógenos pode modificar a dinâmica complexa que ocorrem durante a interação entre um host e um patógeno. Este estudo estabelece que mAbs mediar proteção em um modelo murino de histoplasmose letal quando injetada por via intraperitoneal, e sugere proteínas candidato para o desenvolvimento de vacinas, como H2B, M antígeno (superfície ...

<table border="1"><tbody><tr><th> Nome de Material </th><th> Companhia </th></tr><tr><td> Cabines de segurança biológica (BSL2) </td><td></td></tr><tr><td> 15 mL tubos cônicos </td><td> Falcon, BD </td></tr><tr><td> HAM médio F-12 </td><td> Gibco </td></tr><tr><td> 37 ° C shaker </td><td></td></tr><tr><td> Vórtice </td><td></td></tr><tr><td> 50 mL tubos cônicos </td><td> Falcon, BD </td></tr><tr><td> 26G1 / 2 agulha </td><td> BD </td></tr><tr><td> Seringa de 10 mL </td><td> BD </td></tr><tr><td> Balões de 250 ml </td><td></td></tr><tr><td> FPLC sistema (cromatografia líquida de rápida proteína) </td><td> GE Helthcare </td></tr><tr><td> Leitor de ELISA </td><td> Biotek </td></tr><tr><td> Anestesia (ketamina e xilazina) </td><td></td></tr><tr><td> Coluna está </td><td></td></tr><tr><td> Corda de nylon </td><td></td></tr><tr><td> Lâmpada de calor </td><td></td></tr><tr><td> 70μm filtros celular </td><td> Falcon, BD </td></tr><tr><td> Agar placas BHI </td><td> Gibco </td></tr></tbody></table>

passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens

O objetivo do uso desta metodologia é 1) para avançar a nossa capacidade de proteger os indivíduos com anticorpo ou vacina para prevenir ou tratar histoplasmose causada pelo fungo Histoplasma capsulatum e 2) para examinar o papel de fatores de virulência como alvo para a terapia. Para gerar mAbs, os camundongos são imunizados, as respostas imunes são avaliados através de um sistema de ELISA em fase sólida desenvolvido em nosso laboratório, ea melhor resposta ratos são selecionados para isolamento de esplenócitos para a fusão com células de hibridoma. Camundongos C57BL / 6 têm sido amplamente utilizados para estudar H. patogênese capsulatum e fornecer o melhor modelo para a obtenção dos dados necessários. A fim de avaliar o papel do mAbs em infecção, os ratos são administrados por via intraperitoneal com um mAb para H. capsulatum ou combinados isotipo controle mAb e infectados por um ou outro intravenosa (iv), intraperitoneal (ip), ou intranasal (in) rotas. Na literatura científica, a eficácia de mAbs para infecções fúngicas em camundongos depende de mortalidade como um ponto final, em conjunto com unidades formadoras de colónias formin (CFU) avaliações em momentos anteriores. Sobrevida (tempo de morte) são necessários estudos em que melhor representam a doença humana. Assim, a eficácia da nossa intervenção não ser adequadamente estabelecida sem curvas de sobrevivência. Isto também é verdade para estabelecer a eficácia da vacina ou teste de mutantes para a virulência. Com histoplasmose, os ratos vão frequentemente de ser energético para mortos durante várias horas. A capacidade de uma intervenção como a administração de um mAb podem, inicialmente, proteger um animal de doença, mas a doença pode recaída que não seria realizado em experimentos de curta UFC. Além de sobrevivência e ensaios de carga fúngica, examinamos as respostas inflamatórias à infecção (histologia, recrutamento celular, as respostas de citocinas). Para a sobrevivência / hora para experiências de morte, os camundongos são infectados e monitoradas pelo menos duas vezes por dia para sinais de morbidade. Para avaliar a carga fúngica, histopatologia, e as respostas de citocinas, os ratos são sacrificados em vários momentos após a infecção. Experiências com animais são realizadas de acordo com as diretrizes do Instituto de Estudos Animal do Albert Einstein College of Medicine.

Watch this Scientific Journal Video about Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens at JoVE.com

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Camundongos C57BL / 6 foram usados ​​para estudar a patogênese Hc e fornecer o melhor modelo. Estamos explorando os benefícios potenciais da imunidade humoral contra este fungo e gerou várias mAbs [a histona H2B e um calor 60kDa proteína de choque] que testamos para sua eficácia protectora após a administração intraperitoneal.

Administração passiva de anticorpos monoclonais contra H. capsulatum E outros patógenos fúngicos

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

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Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

11.8K visualizações.  Albert Einstein College of Medicine. Camundongos C57BL / 6 foram usados ​​para estudar a patogênese Hc e fornecer o melhor modelo. Estamos explorando os benefícios potenciais da imunidade humoral contra este fungo e gerou várias mAbs [a histona H2B e um calor 60kDa proteína de choque] que testamos para sua eficácia protectora após a administração intraperitoneal.

Vídeo: Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

A infecção de embriões de zebrafish com patógenos bacterianos intracelulares

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Imunologia e Infecção

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformação Biolistic de uma fluorescente Tagged Gene para o oportunista fúngica Pathogen<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Geração de anticorpos monoclonais murinos pelo hibridoma Tecnologia

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Geração de variantes de fuga de neutralizar os anticorpos monoclonais de vírus da gripe

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Geração dos anticorpos monoclonais humanos discriminativa de rara B antígeno-específicas células circulantes no sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

Geração de anticorpos monoclonais contra produtos naturais

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

Ensaio opsono-adesão para avaliar anticorpos funcionais no desenvolvimento de vacinas contra bacillus anthracis e outros patógenos encapsulados

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...